Our research is focused on understanding the effects of environmental chemicals on mammary gland (MG) development, function, and cancer susceptibility. We have expanded this focus area in recent years to include the effect of environmental chemicals on breast cancer risk factors such as puberty timing, obesity, and lactation. The use of animal models for human disease allows us to evaluate different routes of exposure, internal dose of the test chemical, as well as a variety of effects in the rodent that are relevant to human health. During the last several years, we have produced numerous important documents that are being used in the risk regulation of brominated flame retardants (DE-71), perfluorooctanoic acid (PFOA) and atrazine (ATR). Flame retardants are currently regulated at the state level, as a federal risk assessment has not been completed yet. Our work on DE-71 in the rat are completed for now, but we have expanded our study of flame retardants this year to include tetrabromobisphenol A (TBBPA) and Firemaster 550. I continue to advise the ongoing NTP studies regarding the mechanisms of action of these high use and emerging flame retardants. In the female rodent offspring, the mammary gland is one of the most endocrine-sensitive end points that we have evaluated following prenatal chemical exposures. Our current studies using volatile organic chemicals (VOCs), bisphenol analogues, perfluorooctanoic acid (PFOA), TBBPA, and arsenic all demonstrate this finding. We have nearly finished studies evaluating mammary effects of safe level vs 42 ppm arsenic in mice, human relevant VOC mixtures in rats, PFOA mechanism of action and strain differences in mice, and two BPA replacement chemicals in mice. I have also finalized my evaluation of the mammary samples from the BPA-CLARITY study. Unfortunately we are not sure we can use the data from the CLARITY study due to technical issues in the study resulting in animals not being collected in the same stage of the cycle and contamination of the controls with BPA. Numerous manuscripts are expected from these other studies. We have made major progress on our work involving investigation of VOCs and their developmental exposure effects on the mammary gland, specifically in males. These compounds are theorized to have a role in male breast cancer diagnoses in men who were born or grew up at the Camp Lejeune military base in NC, USA. We have nearly finished dosimetry studies in the pregnant and lactating rat and her offspring in conjunction with our CDC collaborators. We have evaluated mammary gland development and carcinogen-induced mammary tumors following prenatal exposure and have prepared two manuscripts on the effects of these compounds in male and female rats. With this study and our mammary gland atlas of Harlan Sprague Dawley rats that will also be published this coming year, we have added significantly to what is known about male rodent mammary gland development, susceptibility to endocrine disruption, and carcinogen-induced mammary cancer. We are working with Melissa Troester, an epidemiologist at UNC, to document normal aging of the male and female mammary gland of mice and rats, in comparison to her human samples. She has shown that in women with breast cancer there is an interruption of the normal aging process, and by using chemically-exposed rodent tissues, we may determine which types of chemicals may predispose populations to increased breast cancer risk via that mode of action. We have nearly finished studies to look at susceptibility to mammary gland hyperplasia in female mice exposed to bisphenol A (BPA), BPAF and BPAS (fluorinated and sulfonated forms)in early life. These studies are all in line with NTP mission or in collaboration with other Federal agencies. Dispositon studies are in their final stages to understand the transfer of these compounds to the developing fetus and the clearance within the pregnant dam. These studies have been slowed due to low access to mass spec time in the NIEHS core facility and in NTPL. Our recent studies involving TBBPA and Firemaster are large collaborative studies where we are either running the study for many PIs to get target tissues of interest (TBBPA) or we are providing a service of isolating and evaluating mammary tissue (Firemaster). Both of these flame retardants seem to act as endocrine disruptors, affecting multiple reproductive tissues, fat cells, and hormone levels. Several papers on these studies are forthcoming or in clearance. We have recently finished performing a blind evaluation of about 70 chemicals identified in Tox21 as activating specific endpoints thought to be linked to an obesogenic response. We are using mouse 3T3-L1 low passage cells to assess adipogenesis and lipogenesis. Gene expression and follow-up studies have begun on the chemicals identified as most interesting, either for their potential to act as a potent obesogen or because they were a false positive or negative. We are working with a team of investigators, including Kris Thayer at US EPA, and Jui-Hua Hseih in BSB,to produce a document on this concept and test other aspects of the chemical response. From the data accumulated in this group effort, we will better understand the utility and shortcomings of the Tox21 data set in predicting obesogens and may be able to develop assays to add to the high throughput methodology. This FY there has been a emergent need to evaluate the effects of uncharacterized perfluoroalkyl acids (PFAS)and related compounds. We have developed a set of in vitro assays (complimented by our colleagues in the in silico and in vivo areas) that will provide us an our collaborators at the EPA with a rapid understanding of which groups of PFAS are targeting tissues known to be affected byt he legacy compounds before them that are no longer produced in the U.S. We are testing nearly 40 of the most common compounds in liver, mammary cells, placenta, fat cells, and kidney to determine bench mark doses across the group. We are also making mixtures for testing in vitro and in vivo from the mass spec data supplied by our EPA collaborators. Our data is needed by the state of NC and the Dept of Defense in the near future to make decisions on new product use, regulation or filtering of water supplies, and potentially health advisories. We will be very busy in this area in FY18.